Saposins, a family of small (˜80 amino acids), heat stable glycoproteins, are essential for the in vivo hydrolytic activity of several lysosomal enzymes in the catabolic pathway of glycosphingolipids (see Grabowski et al. (1990) Crit. Rev. Biochem. Mol. BioI. 25:385-414; Furst et al. (1992) Biochim. Biophys. Acta. 1126:1-16; Kishimoto et al. (1992) J Lipid Res. 33:1255-1267). Four members of the saposin family (A, B, C, and D) are proteolytically hydrolyzed from a single precursor protein, prosaposin (see Fujibayashi et al. (1985) Am. J Hum. Genet. 37:741-748; O'Brien et al. (1988) Science 241:1098-1101; Rorman et al. (1989) Genomics 5:486-492; Nakano et al. (1989). J Biochem. (Tokyo) 105: 152-154; Reiner et al. (1989) J Mol. Neurosci. 1:225-233; herein incorporated by reference. The complete amino acid sequences for saposins A, B, C, and D have been reported as well as the genomic organization and cDNA sequence of prosaposin (see Fujibayashi et ai. (1985) Am. J Hum. Genet. 37:741-748; O'Brien et ai. (1988) Science 241:1098-1101; Rorman et al. (1989) Genomics 5:486-492).
Saposins are defined as sphingolipid activator proteins or coenzymes. Structurally, saposins A, B, C, and D have approximately 50-60% similarity including six strictly conserved cysteine residues (see Furst et al. (1992) Biochim. Biophys. Acta 1126: 1-16) that form three intradomain disulfide bridges whose placements are identical (see Vaccaro et al. (1995) J. Biol. Chem. 270:9953-9960). All saposins contain one glycosylation site with conserved placement in the N-terminal sequence half, but glycosylation is not essential to their activities (see Qi et al. (1998) Biochemistry 37:11544-11554 and Vaccaro et al. (1995) J. Biol. Chem. 270:30576-30580, herein incorporated by reference in their entirety).
All saposins and saposin-like proteins and domains contain a “saposin fold” when in solution. This fold is a multiple α-helical bundle motif, characterized by three conserved disulfide structures and several amphipathic polypeptides. Despite this shared saposin-fold in solution, saposins and saposin-like proteins have diverse in vivo biological functions in the enhancement of lysosomal sphingolipid (SL) and glycosphingolipid (GSL) degradation by specific hydrolases. Because of these roles, the saposins occupy a central position in the control of lysosomal sphingolipid and glycosphingolipid metabolisms (see Kishimoto et al. (1992) J. Lipid Res. 33:1255-1267; Fujibayashi et al. (1985) Am. J. Hum. Genet. 37:741-748; O'Brien et al. (1988) Science 241:1098-1101, herein incorporated by reference). In addition, saposins participate in the fusion and destabilization of acidic phospholipid vesicles (see Vaccaro et al. (1994) FEBS Letters 349:181-186, herein incorporated by reference).
Saposin C is required for optimal hydrolysis of glucosylceramide by acid β-glucosidase (Gcase, EC 3.1.2.45) in vivo and in vitro. Also, saposin C induced fusion toward phosphatidylserine containing vesicles has been observed by electron microscopy (see Vaccaro et al. (1994) FEBS Letters 349:181-186, herein incorporated by reference). Further, saposin C has the general property of lipid membrane binding activity or plasma membrane affinity. Saposins associate with lipid membranes by embedding into the outer leaflets. The H-1 and H-5 helices are integral to this process, suggesting that proper membrane interaction of saposin C affects its specificity and activity. In addition, saposin C induces structural changes of the membrane. The dynamic processes of saposin interactions with planar phospholipid bilayers have been visualized in real time using atomic force microscopy (see Qi et al. (2001) J. Biol. Chem. 276:27010-27017 and You et al. (2001) FEBS Lett. 503:97-102, herein incorporated by reference).
Phospholipid asymmetry is a well-known characteristic of mammalian plasma membranes. The outer leaflet of the lipid bilayer is rich in choline-phospholipids, whereas aminophospholipids are preferentially in the inner leaflet (Bevers et al. (1998) Lupus Suppl. 2:S126-S131). Phosphatidylserine (PS) and phosphatidylethanolamine (PE) reside almost exclusively in the inner leaflet, and phosphatidylcholine (PC) and sphingomyelin are enriched in the outer leaflet. Phospholipid asymmetry might be a general property of all cells (Woon et al. (1999) Cell Calcium 25(4):313-320). The plasma membrane phospholipid asymmetry is maintained through a variety of mechanisms, including aminophospholipid translocases and phospholipid scramblases (U.S. Patent Application No: 20020081698).
In general, tumor or cancer cells grow rapidly in comparison to normal cells. These abnormal cells produce a significant amount of protons primarily by generating lactic acid during glycolysis or by generating carbon dioxide during respiration due to a fast metabolic rate. Therefore, the surrounding sites of these cells and tissues are usually found to be more acidic than those of cells with a normal growth rate.
Squamous cell carcinomas (SCCs) of the skin are one of the most common skin cancers associated with a substantial risk of metastasis (Alam et al. (2001) N. Engl. J. Med. 344:975-983, herein incorporated by reference). Cancers of the skin are classified into two categories, melanoma and non-melanoma skin cancers (NMSC). According to the estimation by the American Cancer Society, more than one million cases of NMSC are found in the United States each year. SCC accounts for approximately 20% of all cutaneous tumors and there are about 200,000 new SCC cases in the United States each year. SCC is the most frequent form of malignant tumor in the transition from the skin to the mucosa and in the mucosa itself (Boni et al. (2002) Neuroendocrinology Letters 23S2:48-51). The current treatments of SCC patients include electrodessication and curettage, excision, cryotherapy, surgical excision, or Mohs' surgery. Appropriate use of electrodessication and curettage, excision, or cryotherapy can eliminate small (<1 cm in diameter), well-defined tumors with a low risk of metastasis. Surgical excision and Mohs' surgery offer the highest rates of cure for patients with high-risk primary or recurrent SCCs. However, these treatments are more costly with the risk of hematoma, seroma, infection, and wound dehiscence.
Thus, development of an effective, low-cost SCC treatment with improved cosmetic outcomes is desirable. It is also of importance to develop an effective, low-cost treatment for other cancer types such as breast and prostate cancers and lymphomas.